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Purpose To investigate the effects of hypoxia inducible factor-2α (HIF-2ot) siRNA on proliferation and chemotherapy sensitivity of breast carcinoma MCF-7.Methods RNA interference was used to silence the expression of HIF-2α in MCF-7 cells.The changes of HIF-2α gene expression were detected by immunocytochemistry and RT-PCR.Under hypoxia environment simulated by CoCl2,MTT assay and flow cytometry (FCM) were used to measure cell growth inhibition rate and cell apoptosis of MCF-7 cells under different dosages of chemotherapeutic agents (5-fluorouracil,adriamycin,and paclitaxel).Results Expression of HIF-2α in MCF-7 were down-regulated by HIF-2α siRNA (P ＜ 0.05).The proliferation inhibition and apoptosis rates were evidently increased after transfection with HIF-2α siRNA (P ＜ 0.05),chemotherapy drug sensitivity was enhanced.Conclusion HIF-2α siRNA can induce the apoptosis and inhibit the proliferation and enhance the sensitivity of breast carcinoma MCF-7 cell line to chemotherapeutic agents.Blocking HIF-2α maybe a very promising strategy for breast carcinoma gene therapy in combination with chemotherapy.
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Objective To investigate the role of hypoxia?inducible factor?2α(HIF?2α) in the expression of tight junction proteins and permeability alterations in rat glomerular endothelial cells (rGENCs) under hypoxia condition. Methods The expressions of the HIF?2α and tight junction proteins such as occludin and ZO?1 of rGENCs were examined after exposed to 5%oxygen at different treatment time periods (0 h, 12 h, 24 h and 48 h). Then lentiviral transfection was used to knock down HIF?2α expression in rGENCs. The cells were split into four groups, including i) control group where rGENCs were cultured under normal oxygen conditions, ii) hypoxia group, iii) negative control group where rGENCs were infected with a negative vector, iv) HIF?2α lentivirus transfection group. Group ii, iii and iv were kept in hypoxic chamber (5% O2, 5% CO2 and 90% N2) for 24 h. The expressions of occludin, ZO?1 and HIF?2α were assessed by Western blotting. The permeability of rGENCs was measured using trans?epithelium electrical resistant (TEER) by Millicell? ERS voltohmmeter. Results With the elongation of hypoxia time, the expression of HIF?2α was increased gradually, while the occludin expression was decreased, there was statistically significance difference in each group (all P0.05). And a dramatic decrease in TEER of hypoxia cells was detected as compare with control cells (P0.05). Conclusion Hypoxia may promote HIF?2α expression, which could increase the permeability of rGENCs by reducing the expression of occludin and ZO?1.
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Objective To investigate the effect of HIF-2a silencing by transfection of siRNA into MG-63 cells un-der hypoxia. Methods HIF-2αexpression level in MG-63 cells under hypoxia was determined by Western Blot. Small in-terfering RNA (siRNA) was used to construct MG-63/siHIF-2α(siHIF-2α)cells and control MG-63/scramble (NC) cells. The expression levels of HIF-2α, Vascular endothelial growth factor (VEGF), p-Erk/ErK and Mcl-1 in MG-63, NC and si-HIF-2αcells was determined by Western Blot. NC and siHIF-2αcells were cultured under hypoxia. Cell viability was as-sessed by MTT assay. Migration was identified by scratch migration assay. Tumor formation was identified by clone formation assay. Nude mouse subcutaneous xenograft model was used to investigate tumor development in vivo. Results Hypoxia im-proved HIF-2αexpression in MG-63 cells in a time-dependent manner (F=2 037.412,P<0.001). HIF-2αexpression un-der hypoxia in siHIF-2αcells was lower than that in NC cells (P<0.01). Cell viability of siHIF-2αcells under hypoxia for 12 h and 24 h were lower than that in NC cells (P<0.05 or P<0.01). The relative width of scratch in siHIF-2αgroup under hypoxia for 12 h and 24 h were larger than that in NC group (P<0.01 or P<0.01). When cell counts reach 1 000-5 000, the clone formation rates of siHIF-2αcells were lower than that in NC cells (P<0.05 or P<0.01). The expression of VEGF, p-Erk/Erk and Mcl-1 protein under hypoxia in siHIF-2αcells was lower than that in NC cells(P<0.01). Tumor sizes, weights and density of siHIF-2α group in nude mice were suppressed compared with those in NC group (P<0.01). Conclusion Blocking HIF-2αsignal pathway warrants its investigation as a potential strategy in osteosarcoma treatment.
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Objective To investigate the location and expression of hypoxia inducible factor (HIF) subunits in the remnant kidney of 5/6 nephrectomy rats. Methods Remnant kidneys were produced in adult male SD rats by 5/6 nephrectomy. The renal function and histopathological changes were evaluated at week 1, 2, 4, 6, 8 and 12 after operation. Tissues of remnant kidneys were collected to detect the location and expression of HIF-1α and HIF-2α by immunohistochemistry staining and Western blotting. The mRNA levels of HIF targeted genes vascular endothelial growth factor (VEGF) and heme oxygenase-1 (HO-1) were determined by RTPCR. Results (1) 5/6 nephrectomy rats underwent one week of acute renal failure at first[Scr (122.8±22.1) μmol/L] and then developed compensative chronic renal failure [(66.0±3.7)-(66.4±8.4) μmol/L], but the level of Scr increased quickly after week 6 [(66.4±8.4)-(127.8±22.7) μmol/L],concomitantly with progressive tubulointerstitial fibrosis in remnant kidney cortex. (2) In cortex, HIF-1α was expressed only in the atrophic and dilated tubular cells while HIF-2α was located in endothelial, interstitial fibroblasts, and vascular smooth muscle cells. The semiquantitative results of imunohistochemistry and Western blotting revealed that HIF-1α and HIF-2α were both gradually up-regulated during the early stage of remnant kidney, peaked at week 4 and 6, and then gradually down-regulated. (3) The mRNA levels of HIF targeted genes VEGF and HO-1 transiently peeked at week 4 and 6, and then decreased gradually. Conclusions The increased stabilization of HIF-αprotein and transcription of HIF targeted genes at the early stage of this model is a compensation reaction towards hypoxia. The mechanism of decreased expression of HIF-α at the end stage of chronic kidney disease deserves further investigation.